JP3125284B2 - Method for measuring hemoglobin in stool and tool for measuring the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring hemoglobin which can determine the presence or absence of hemoglobin and the concentration of hemoglobin simultaneously with the collection of feces when measuring hemoglobin contained in feces. The present invention relates to a measuring tool to be used.

[0002]

2. Description of the Related Art For the purpose of early detection of diseases of the digestive system such as colon cancer and stomach cancer, a method of detecting occult blood in feces has been implemented as screening means. In particular, immunological methods using antibodies to human hemoglobin,
Recently, it is becoming mainstream in place of biochemical methods because it can be detected with high sensitivity without the need for dietary restriction. Testing methods for achieving these tests more easily (JP-A-59-182667, JP-A-61-228951, etc.) and diagnostic instruments therefor (JP-A-61-228951) Have been proposed.

[0003]

SUMMARY OF THE INVENTION The above-mentioned Japanese Patent Application Laid-Open No. 59-18 / 1984
No. 2367 discloses a method for detecting hemoglobin in feces, which comprises a collecting device made of a polymer material and a liquid container containing a buffer solution previously adjusted to prevent the activity of digestive enzymes in feces. Using a container, hemoglobin in feces is physically adsorbed and separated on the surface of the collection device,
The hemoglobin is specifically detected and measured using anti-human enzyme-labeled hemoglobin. However, the test method using the collection container requires that hemoglobin be physically adsorbed on the surface of the collection device by a buffer contained in a liquid container, and thereafter, hemoglobin in feces by a known enzyme immunoassay. Since the measurement requires a very complicated operation, the test has not always been simplified because the reagents used for the enzyme immunoassay have to be prepared separately. The method for detecting hemoglobin disclosed in Japanese Unexamined Patent Publication No. 61-228951 also has almost the same problems.

In view of such circumstances, the present invention provides a method for measuring hemoglobin in feces, which can measure hemoglobin in feces in a short time and with high sensitivity by a very simple operation, and a measuring tool therefor. It is intended for that purpose.

[0005]

In order to achieve the above object, a method of measuring hemoglobin in stool according to the present invention comprises a container body holding a developing solution at an upper part and a substrate solution at a lower part by a partition wall capable of piercing. A stool serving as a sample was applied to the sampling member by using a deployment member having a capillary action, and a measurement member in which a sampling member for collecting stool having a capillary action was engaged or held directly or via an absorption pad. After that, the sampling member is inserted into the container body, the developing solution is punctured into the partition wall of the developing solution chamber by the sampling member, and the developing solution is guided to the developing member via the sampling member, and the enzyme-labeled anti-hemoglobin antibody carried on the developing member is used. It reacts with hemoglobin in the stool, and then punctures the partition wall of the substrate liquid chamber with a sampling member and sends the substrate liquid to the developing member, and then the anti-hemog is applied to the developing member. Reagent consisting bottle antibody is characterized in that to determine the hemoglobin in feces in the detection zone which is carried.

More specifically, a container body holding a developing solution such as a physiological saline solution at an upper portion thereof and a substrate solution for coloring, emitting or fluorescent light at a lower portion thereof by puncturing partition walls,
A proximal end portion of a rod-shaped sampling member for collecting stool having a capillary action and having a capillary action formed of a fibrous structure on one of the absorption pads while a deployment member having a capillary action is provided between the absorption pads arranged at predetermined intervals. Using a measurement member engaged and held, a stool serving as a sample is applied to the tip of the sampling member, and after removing excess stool, the tip of the sampling member is inserted into the container body, The developing solution is guided to the developing member through the sampling member and the absorption pad by puncturing the partition wall of the developing solution chamber at the tip of the sampling member, and the enzyme-labeled anti-hemoglobin antibody applied to the developing member reacts with hemoglobin in the stool. Thereafter, the partition wall of the substrate liquid chamber is punctured at the tip of the sampling member to supply the substrate liquid to the developing member, and the colorimetric method or light emission is performed in the detection zone formed on the developing member. Or it is characterized in determining the hemoglobin in feces by fluorescence.

On the other hand, a measuring tool used for the method of measuring hemoglobin in stool is provided with two piercing partitions below the inside, a developing liquid chamber at the upper part, a substrate liquid chamber at the lower part, and Engaging and holding the base end of the sampling member for collecting feces directly or via an absorbing pad with a developing member having a capillary action, and enzymatically labeled anti-hemoglobin at a predetermined interval from the sampling member side to the developing member It is characterized by comprising an antibody and a reagent composed of an anti-hemoglobin antibody, and a measuring member formed so that at least a portion supporting the reagent can be visually observed from the outside.

[0008] More specifically, 2 which can be punctured downward inside.
A container main body having a partition wall provided at a predetermined interval with an upper portion serving as a developing liquid chamber and a lower portion serving as a substrate liquid chamber, and a tubular shape which can be freely inserted and removed in contact with the inner peripheral surface of the container main body. An absorbent pad is disposed at a predetermined interval in the body, a deployment member having a capillary action is laid between the absorbent pads, and a rod-shaped sampling member for collecting stool made of a fibrous structure is provided on one of the absorbent members. Enzyme-labeled anti-hemoglobin antibody and a reagent consisting of an anti-hemoglobin antibody are dried and frozen on the developing member at a predetermined interval from the sampling member side to support the developing member, and at least the reagent And a measuring member formed so that the portion carrying the can be viewed from the outside.

Hereinafter, an example of the fecal hemoglobin measuring device of the present invention will be described in detail with reference to the accompanying drawings. The fecal hemoglobin measuring device 1 of the present invention comprises a container body 2
And a measuring member 13. The container body 2
Two partition plates 3, 4 each having a through hole formed in the center at the lower part inside a transparent bottomed cylindrical body made of a synthetic resin are arranged at a predetermined interval, and the upper part is a developing liquid chamber 5, The lower part is the substrate liquid chamber 6, and the partition walls 7, 8 are capable of piercing the through holes of the partition plates 3, 4.
And a cylindrical guide member 9 protruding from the upper surface of the partition plate 3 of the developing liquid chamber 5 so as to surround the through hole.

In the container body 2, a concave guide groove 10 is formed in a part of the side wall from the opening end to the vicinity of the partition plate 3 of the developing liquid chamber 5 in parallel with the axis. A plurality of engaging holes 11a, 11b, and 11c are provided at predetermined intervals, and a notch 12 with a predetermined width is formed on a side wall facing the guide groove 10 from an opening end to a bottom.
Are formed.

The container body 2 has an opening at both ends and a partition plate 3 below to facilitate molding and fill the developing liquid chamber 5 and the substrate liquid 6 with the developing liquid and the substrate liquid, respectively. A container member 2a, a container member 2b having a partition plate 4 below which is screw-connected to a lower opening of the container member 2a, both ends of which are open, and a bottomed container screw-connected to a lower opening of the container member 2b And a member 2c.

The measuring member 13 has absorption pads 14 and 15 disposed at predetermined intervals in a cylindrical body which can be inserted and removed in a state of being in contact with the inner peripheral surface of the container body 2. A deployment member 1 having a capillary action between the absorption pads 14 and 15
6 and engages and holds the base end of a rod-shaped sampling member 17 for collecting stool made of a fibrous structure on one of the absorbent pads 14, and projects with the guide groove 10 of the container body 2. A strip 18 is formed on the outer peripheral wall of the cylindrical body,
8 has an engaging hole 11 formed in the guide groove 10 at the tip thereof.
a, 11b, and 11c are provided, and a plurality of through holes 20a, 20b, and 20c are provided on a side wall portion facing the ridge member 18 at predetermined intervals on the same line. It is a thing. In addition, the deployment member 1
The sampling member 17 may be provided directly at the base end of the sixth member.

Thus, the sampling member 17 is
For example, a polyester fiber bundle is bonded and integrated with an adhesive, and is formed of a fiber structure having a capillary action formed into a round bar having a diameter of about 2 mm. An outer peripheral surface coated with a vinyl chloride resin film to have a smooth and non-water-absorbing surface can be used.

Each of the absorbent pads 14, 15 may be formed by laminating filter paper to form a predetermined thickness, a sponge,
Alternatively, the developing member 16 is made of a cotton body, and is made of a band-shaped filter paper made of cellulose or glass fiber having a predetermined width and thickness and having a capillary action.

The developing member 16 is provided with an enzyme-labeled anti-hemoglobin antibody (hereinafter referred to as "enzyme-labeled antibody") at a site corresponding to the through-hole 20a formed in the cylindrical body.
And a performance confirmation reagent for confirming the performance of the developing solution and the substrate solution filled and sealed in the container main body 2 are freeze-dried and carried in the portion corresponding to the through hole 20c. , A preliminary zone 21, a detection zone 22, and a performance confirmation zone 23 are formed from the sampling member side.

As the enzyme-labeled antibody, for example, an enzyme such as alkaline phosphatase, peroxidase, β-D-galactosidase, and an anti-human hemoglobin polyclonal antibody, an anti-human hemoglobin monoclonal antibody or a degradation product thereof are bound by a known method. As a reagent in the detection zone, an anti-human hemoglobin polyclonal antibody, an anti-human hemoglobin monoclonal antibody or a decomposition product thereof is bound to a solid phase such as polystyrene or polymethacrylate gelatin as an antibody. A hemoglobin antibody-bound solid phase can be mentioned, but the antibody can also be used by directly engaging the antibody with a developing member without using a solid phase. The enzyme-labeled antibody and the anti-human hemoglobin antibody used as the detection reagent are preferably antibodies having different hemoglobin recognition sites. Furthermore, examples of the performance confirmation reagent include anti-enzyme antibody-bound latex such as anti-peroxidase antibody-bound latex.

On the other hand, the developing liquid chamber 5 formed in the container main body 2 is filled with a developing liquid, and the substrate liquid chamber 6 is filled with a substrate liquid, respectively, and is sealed by puncturing partitions 7 and 8. However, as a developing solution, phosphate buffered saline,
Acetate buffered saline, Tris-HCl buffer, and a surfactant (Tween 20; Triton X-100;
PVP; PVA, etc.) can also be added.

Examples of the substrate solution include a solution containing a chromogenic substrate, a fluorescent substrate, a luminescent substrate and a substrate. Examples of the chromogenic substrate include 3,3 ′, 5,5′-tetramethylbetidine (TMB) and 2,2′-amino-di (3-
Ethylbenzthiazoline) -6'-sulfonic acid (ABT
S), compounds for peroxidase (POD) such as diaminobenzidine (DAB) and for alkaline phosphatase such as BCIP. As the fluorescent substrate, 4-methylum beryphenyl phosphate (4M
UP) or for alkaline phosphatase, 4-methylum beryphenyl / β-D-galactoside (4MUG)
And other compounds for β-D-galactosidase.

As the luminescent substrate, 3- (2'-spiroadamantane) -4-methoxy-4- (3 "-phosphoryloxy) phenyl-1,2-dioxetane (AMPP)
D) and the like, or for 3- (2′-spiroadamantane) -4-methoxy-4- (3 ″ -β-D
-Galactopyranosyl) phenyl-1,2-dioxetane (AMGPD) and other compounds for β-D-galactosidase and peroxidases such as luminol and isoluminol. Examples of the solution containing the substrate include an acetate buffer, a borate buffer, a Tris-HCl buffer, a diethanolamine buffer, and the like.

The stool hemoglobin measuring device 1 composed of the container body 2 and the measuring member 13 is used by the following method. Normally, before the use of the hemoglobin measuring device 1 of the present invention, as shown in FIG. 2, the distal end of the sampling member 17 constituting the measuring member 13 And the distal end of the cylindrical body is in contact with the inner peripheral wall of the container body 2 and the measuring member 13
The projection member 19 of the ridge 18 is held in a state of being engaged with the first through hole 11 a provided in the guide groove 10 formed in the container body 2. In this state, the developing liquid is filled in the developing liquid chamber 5 of the container body 2 and the substrate liquid is filled and sealed in the substrate liquid chamber 6, but the projecting member 19 and the through hole 11a are sealed.
The tip of the sampling member 17 is not at a position where the partition wall 7 of the developing liquid chamber 5 is punctured by the engagement with the developing member.

In use, the test subject gently presses the projection member 19 inward to use the container body 2 and the measurement member 1.
3 is released, and then the measuring member 13 is detached from the container body 2. Next, the distal end of the sampling member 17 is pierced into the stool by holding the cylindrical body of the measuring member 13, and excess stool is wiped off with a cloth or tissue paper. When the strip 18 is gradually pressed into the container body 2 while engaging the guide groove 10 of the container body 2, the measuring member 13 advances downward beyond the first through-hole 11a, as shown in FIG. The tip of the sampling member 17 punctures the diaphragm 7 formed on the partition plate 3, the projection member 19 of the measurement member 17 engages with the second through-hole 11 b, is fixed at that position, and enters the developing liquid chamber 5. The filled developing liquid is sent to the developing member 16 via the sampling member 17 and the absorbing pad 14.

At this time, the hemoglobin in the stool is also sent to the developing member 16 together with the developing solution, and reacts with the enzyme-labeled antibody carried in the preliminary zone on the absorption pad 14 side of the developing member 16 to carry the detection reagent. The detection pad and the performance confirmation reagent are carried and the absorption pad 15 passes through the performance confirmation zone.
In this state, no reaction occurs in the detection zone and the performance confirmation zone.

Next, when the developing liquid in the developing liquid chamber 5 becomes empty, the measuring member 13 is further pressed into the container main body 2.
The tip end of the sampling member 17 punctures the partition wall 8 provided on the partition plate 4, and the projection member 19 of the ridge 18 provided on the measurement member 13 engages with the third through hole 11c to maintain the state. Therefore, the substrate liquid in the substrate liquid chamber 6 flows to the developing member 16 via the sampling member 17 and the absorption pad 14, and reacts with the detection reagent carried in the detection zone to generate color, fluorescence, or emit light, and at the same time, to the performance confirmation zone. Reacts with the performance confirmation reagent.

That is, if the substrate liquid is a chromogenic substrate, it develops color in the detection zone. Therefore, the color development state is determined visually or by using a colorimeter, reflected light meter or the like to determine negative or positive. Is a fluorescent substrate, a fluorometer is used in the inspection zone, and a luminescent substrate solution is judged to be negative or positive using an instant film, a photo counter, a photodiode, or the like.

When the measuring member 13 is inserted into the container main body 2, the through holes 20a, 20b, and 20c provided in the measuring member 13 are located in the notches 12 formed in the side wall of the container main body 2, respectively. Since it is configured, the container main body 2 does not interfere with visual observation or the like.

[0026]

According to the method of measuring hemoglobin in stool of the present invention, a sampling member from which stool hemoglobin is collected by piercing is inserted into a container body filled with a developing solution on the upper side and a substrate liquid on the lower side and sealed. In a very simple operation, the developing solution is first absorbed by the sampling member and then the substrate solution is supplied to the developing member via the absorption pad, and the hemoglobin concentration in the feces is reduced at the same time as the sampling of hemoglobin in the stool. It can be measured.

The measuring tool used in the above-mentioned measuring method comprises a container body which can be filled and sealed with a substrate solution and a developing solution, a sampling member which can be inserted into the container body and collects hemoglobin in stool, and an enzyme label. Since it is composed of a measuring member consisting of a developing member carrying an antibody and a test reagent, it is possible to reliably collect hemoglobin in the stool and measure the collected hemoglobin with high sensitivity using an extremely simple tool. it can.

[0028]

EXAMPLES The method of measuring hemoglobin in stool and the measuring tool of the present invention will be specifically described below with reference to examples.
As shown in FIG. 5, 40 μl of a peroxidase-labeled anti-human hemoglobin mouse IgG solution (1 μg / ml and 70 mM ascorbic acid, pH7. 0), and 40 μl of anti-human hemoglobin IgG-bound latex as a test reagent was spotted on the detection zone 22 at about 25 mm from the left end, and freeze-dried to measure hemoglobin about 50 mm long and about 7 mm wide. Was obtained.

On the other hand, the developing solution (0.05% Tween-20, 0. 0%) is stored in the developing solution chamber 5 of the container body 2 shown in FIGS.
02M phosphate buffered saline, pH 7.0)
μl, and the substrate liquid chamber 6 (20 mM trisodium citrate, 125 mM NaCl, 0.2% T
500 μl (ween 80, 0.4 mM TMB, 0.005% hydrogen peroxide solution) were filled, and the through holes of the partition plates 3 and 4 were heat-sealed with an aluminum laminate film, respectively, and sealed. For the sake of explanation, these aluminum laminated films are the same as the partition walls denoted by reference numerals 7 and 8 in the figure.

On the other hand, the developing member 16 obtained above is laid between the absorbent pads 14 and 15, leaving only a predetermined length at both ends of the fibrous structure, and coating the remaining outer peripheral portion with a vinyl chloride resin film. About 2mm in diameter and about 5 in length
The base member of the 5 mm sampling member 17 was engaged with and held by the absorbing pad 14 to form the measuring member 13.

Thereafter, 50 μl of stool extract is spotted on the tip of the sampling member 17, and then the sampling member 17 is inserted into the container main body 2, and the through hole of the partition plate 3 is inserted at the tip of the sampling member 17. The heat-sealed aluminum laminated film 7 was punctured, and the developing solution was suction-developed on the upper part. The reaction of the developing member 16 in this state is as shown in FIG.

Then, when the developing solution in the developing solution chamber 5 is empty, the sampling member 17 is further pressed downward to pierce the aluminum laminate film 8 at the tip thereof.
The substrate solution was suction-developed on the upper portion, and after 15 minutes, a spot formed at the position of the detection zone 22 in FIG. 7 was visually judged, and the color development amount was measured by a colorimeter. FIG. 7 shows the deployment member 16 in this state.

[0033]

According to the method for measuring hemoglobin in stool of the present invention, a container body filled with a developing solution in the upper part and a substrate liquid in the lower part and sealed, a sampling member for collecting hemoglobin in the stool, an enzyme-labeled antibody and a test This is performed using a measuring member composed of a developing member carrying a reagent, and a sampling member is collected from which hemoglobin in feces is collected by piercing the container body. Liquid is absorbed by the sampling member,
With a very simple operation of supplying this to the developing member via the absorption pad, the hemoglobin concentration can be measured in a short time simultaneously with the sampling of hemoglobin in the stool.

The measuring instrument used in the above-mentioned measuring method comprises a container body which can be filled and sealed with a substrate solution and a developing solution, a sampling member which can be inserted into the container body and collects hemoglobin in stool, and an enzyme label. Since it is composed of a measuring member consisting of a developing member carrying an antibody and a test reagent, it is possible to construct a measuring tool for hemoglobin in stool with extremely simple members, which is necessary for measuring hemoglobin in stool. Since all the necessary members and liquids are made compact, anyone can easily and easily perform the measurement by using this measuring tool, and a low-cost measuring tool can be provided.

[Brief description of the drawings]

FIG. 1 is an exploded front view showing an example of a tool for measuring hemoglobin in feces of the present invention.

FIG. 2 is a sectional view of the measuring tool in an unused state.

FIG. 3 is a cross-sectional view showing a state in which a developing solution is suction-developed on an upper portion.

FIG. 4 is a cross-sectional view showing a state in which a substrate liquid is suction-developed on an upper portion.

FIG. 5 is a front view of a developing member showing a developing state of a developing liquid by suction;

FIG. 6 is a front view of the developing member showing a state in which the developing liquid has been expanded.

FIG. 7 is a front view of the developing member at the end of the development of the substrate liquid.

[Description of Signs] 1 Measurement tool 2 Container body 5 Developing liquid chamber 6 Substrate liquid chamber 7 Partition 8 Partition 10 Guide groove 13 Measurement member 14 Absorbing pad 15 Absorbing pat 16 Deploying member 17 Sampling member 18 Ridge 21 Preliminary zone 22 Detection Zone 23 Performance confirmation zone

Claims (6)

(57) [Claims] 1. A container body holding a developing solution at an upper part by a partition wall capable of piercing a substrate solution at a lower part, and a sampling member for collecting stool having a capillary action directly or absorbed by a developing member having a capillary action. Using a measuring member engaged and held via a pad, applying a stool serving as a sample to the sampling member, inserting the sampling member into the container body, and puncturing a partition of the developing liquid chamber with the sampling member. The developing solution is guided to the developing member via the sampling member, and the enzyme-labeled anti-hemoglobin antibody carried on the developing member is allowed to react with hemoglobin in the stool. Thereafter, the sampling member punctures the partition wall of the substrate liquid chamber. The substrate liquid is supplied to the developing member, and the hemoglobin in the feces is determined in the detection zone in which the reagent comprising the anti-hemoglobin antibody is carried on the developing member. And a method for measuring hemoglobin in stool. 2. A container body holding a developing solution such as a physiological saline solution at an upper portion thereof and a substrate solution for coloring, emitting or fluorescent light at a lower portion thereof by a piercable partition wall, and an absorption pad arranged at a predetermined interval. A measuring member having a deployment member having a capillary action interposed therebetween, and a base member of a rod-shaped sampling member for collecting feces having a capillary action engaged and held on one of the absorption pads is used. Then, a stool serving as a sample is applied to the tip of the sampling member, and after removing excess stool, the tip of the sampling member is inserted into the container body, and the partition of the developing liquid chamber is separated by the tip of the sampling member. The puncture guides the developing solution to the developing member via the sampling member and the absorption pad, and reacts the enzyme-labeled anti-hemoglobin antibody applied to the developing member with hemoglobin in the stool. After that, puncture the partition wall of the substrate liquid chamber at the tip of the sampling member and feed the substrate liquid to the developing member, and determine the hemoglobin in the stool by a color development method, a luminescence method, or a fluorescence method in the detection zone formed on the development member. A method for measuring hemoglobin in stool. 3. A container body having two piercable partition walls provided below the inside thereof, an upper part serving as a developing liquid chamber, and a lower part serving as a substrate liquid chamber, and a developing member having a capillary action as a sampling member for collecting feces. Engage and hold the base end directly or via an absorption pad, and carry an enzyme-labeled anti-hemoglobin antibody and a reagent comprising an anti-hemoglobin antibody at a predetermined interval from the sampling member side to the developing member, A measuring instrument for measuring hemoglobin in stool, comprising: a measuring member formed so that a portion supporting the reagent is externally visible. 4. A container body in which two punctures are provided at a predetermined interval below the inside of the container, and an upper part is a developing liquid chamber and a lower part is a substrate liquid chamber, and an inner peripheral surface of the container body. Absorbing pads are disposed at predetermined intervals in a cylindrical body that can be inserted and removed in a state where the absorbing pads are in contact with each other, and a deploying member having a capillary action is provided between the absorbing pads, and fibers are attached to one of the absorbing members. A reagent comprising an enzyme-labeled anti-hemoglobin antibody and an anti-hemoglobin antibody at a predetermined distance from the sampling member side to the deployment member, with the base end of a rod-shaped stool sampling sampling member made of a structure engaged and held. And a supporting member formed by drying and freezing, and a measuring member formed so that at least a portion supporting the reagent is visible from the outside. 5. The container body is provided with two partition plates formed with a through hole in the center at a lower portion of the inside of the container body at a predetermined interval, an upper portion serving as a developing liquid chamber, and a lower portion serving as a substrate liquid chamber. The through hole of each partition plate is sealed by a pierceable partition, and a cylindrical guide member is provided on the upper surface of the partition plate of the developing liquid chamber so as to surround the through hole. Item 4. The tool for measuring hemoglobin in stool according to Item 4. 6. The container body has a concave guide groove formed in a part of the side wall from the opening end to the vicinity of the partition plate of the developing liquid chamber in parallel with the axis, and a required interval is provided between the guide grooves. A plurality of engaging holes, a ridge that engages with the guide groove is formed on the outer peripheral wall of the cylindrical body that constitutes the measurement member, and a guide groove is formed at the tip of the ridge. The stool hemoglobin measurement according to claim 4 or 5, wherein a projection member is provided for engaging with the engagement hole formed, and the measurement member is locked to a predetermined portion of the container body as necessary. Tools.